Molecules and methods for inhibiting shedding of KIM-1

ABSTRACT

Disclosed are antibodies that inhibit proteolytic release of a soluble KIM-1 polypeptide from KIM-1 expressing cells. Also disclosed are methods of using the antibodies to inhibit shedding of the KIM-1 polypeptide.

This application is a continuation of International Patent ApplicationNo. PCT/US02/17402, filed May 31, 2002, which claims priority to U.S.Provisional Application Nos. 60/295,449, filed Jun. 1, 2001, expired,and 60/295,907, filed Jun. 4, 2001, expired.

FIELD OF THE INVENTION

The invention relates to antibodies that bind to polypeptides expressedin injured or diseased kidney cells, as well as to methods forproduction and the use of such antibodies.

BACKGROUND OF THE INVENTION

The kidney injury-molecule-1 (“KIM-1”) gene was identified as a genewhose expression is upregulated in post-ischemic rat kidney cells ascompared to the expression of the gene in non-injured rat kidney cells.The KIM-1 gene encodes a type I cell membrane glycoprotein. Two forms ofthe gene have been described in humans. One form is named KIM-1(a) andis 334 amino acids in length. The second form is named KIM-1(b) and is359 amino acids in length. The two human homologues are identicalthroughout their 323 amino terminal amino acid sequences but differ insequence in their carboxy terminal amino acids. The KIM-1 gene isexpressed in dedifferentiated proximal tubular epithelial cells indamaged regions. High level expression is observed in the S3 segment ofthe proximal tubule in the outer stripe of the outer medulla. Thisregion is highly susceptible to damage as a result of ischemia ortoxins.

The amino terminal region of the KIM-1 protein includes theextracellular portion of the KIM-1 protein. This region includes asix-cysteine immunoglobulin-like domain and a T/SP rich domaincharacteristic of mucin-like O-glycosylated proteins.Immunoglobulin-like domains have been widely implicated in mediatingprotein-protein interactions, particularly at the cell surface wherethey are responsible for cell-cell and cell-extracellular matrixinteractions.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that monoclonalantibodies raised against the human KIM-1 extracellular domain caninhibit proteolytic release (shedding) of a soluble KIM-1 polypeptidefrom the membrane-associated form of the KIM-1 protein.

In general, the invention features an antibody, antibody derivative, orantigen-binding polypeptide that inhibits proteolytic release of asoluble KIM-1 polypeptide from KIM-1-expressing cells. The antibody canbe a monoclonal antibody or a polyclonal antibody. The antibody can be ahumanized monoclonal antibody or a fully human monoclonal antibody. Theantibody can include, e.g., an IgG polypeptide.

The antibody binds to the extracellular domain of a full length KIM-1polypeptide. In some embodiments the antibody binds to an epitopelocated within the amino acid sequence SSDGLWNNNQTQLFLEHS (SEQ ID NO:1)in the extracellular domain of a KIM-1 polypeptide.

Also provided by the invention is a conjugate that includes aproteolysis-inhibiting KIM-1 antibody, antibody derivative or antigenbinding polypeptide linked to a detectable label. The detectable labelcan be, e.g., a radiolabel or a fluorescent label.

The invention also includes a conjugate or fusion polypeptide thatincludes a KIM-1 proteolysis-inhibiting antibody, antibody derivative,or antigen-binding polypeptide and a toxin moiety.

Also within the invention is an antibody that has the same epitopespecificity as the antibody produced by the hybridoma deposited in theATCC under Accession No. PTA-3350.

The invention additionally provides an antibody that crossblocks bindingof the antibody produced by the hybridoma deposited in the ATCC underAccession No. PTA-3350. In some embodiments, the antibody is produced bythe hybridoma deposited with ATCC as Accession No. PTA-3350. Alsofeatured by the invention is a nucleic acid encoding the monoclonalantibody produced by the hybridoma.

The invention also features the hybridoma deposited with ATCC underAccession No. PTA-3350.

The invention features a composition that includes the herein describedKIM-1 proteolysis-inhibiting antibody, antibody derivative, orantigen-binding polypeptide and a pharmaceutically acceptable carrier.

The invention also includes a method of inhibiting release of a solubleform of a KIM-1 polypeptide from a cell. The method includes contactinga cell expressing a KIM-1 cell surface polypeptide with an effectiveamount of a KIM-1 proteolysis-inhibiting antibody, antibody derivative,or antigen-binding polypeptide. The cell can be, e.g., a renal cell. Insome embodiments, the renal cell is a renal cancer cell.

The cell can be provided in vitro or in vivo. Preferably, the effectiveamount of antibody is between about 0.1 and 100 mg/kg, more preferablybetween about 0.5 and about 50 mg/kg, and still more preferably betweenabout 1 and about 20 mg/kg. When the cell is provided in vivo, theeffective amount of antibody can be administered by intravenous infusioninto a subject during an infusion period of 1–6 hours. In someembodiments, the soluble form of the KIM-1 polypeptide includes thepolypeptide sequence VKVGGEAGP (SEQ ID NO:2). Data from Example 3revealed that a soluble form of KIM-1 which was released into theextracellular milieu by proteolytic cleavage at a site proximal to thetransmembrane domain included the amino acid sequence given by SEQ IDNO:2.

Also provided by the invention is a method of inhibiting proteolyticshedding of a KIM-1 fragment by contacting a cell expressing a KIM-1polypeptide comprising the fragment with an effective amount of a KIM-1proteolysis-inhibiting antibody, antibody derivative, or antigen-bindingpolypeptide.

The invention also includes a method of treating or preventing renaldisease or injury. The method includes administering to a mammal, e.g.,a human, in need thereof a KIM-1 proteolysis-inhibiting antibody,antibody derivative, or antigen-binding polypeptide. An example of arenal disease that can be treated is renal cancer, e.g., renalcarcinoma.

As used herein, the term “antibody” refers to an immunoglobulin moleculeand immunologically-active portions of the immunoglobulin molecule,i.e., a molecule that contains an antigen binding site thatspecifically-binds (immunoreacts with) an antigen. Such antibodiesinclude, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab and F(ab′)₂ fragments, and an Fab expression library.

As used herein, the term “derivative” refers to a molecule that containseither additional chemical moieties that are not normally a part of themolecule or contains less moieties than are normally a part of themolecule. The addition or subtraction of moieties may improve themolecule's solubility, absorption, or biological half-life or maydecrease the toxicity of the molecule.

As used herein, the term “antigen-binding polypeptide” refers to anantibody fragment, variant, analog, or chemical derivative that retainsthe antigen-binding properties of the antibody.

As used herein, the term “monoclonal antibody” (MAb) refers to apopulation of antibody molecules that contain only one molecular speciesof antibody molecule consisting of a unique light chain gene product anda unique heavy chain gene product. In particular, the complementaritydetermining regions (CDRs) of the monoclonal antibody are identical inall the molecules of the population. MAbs thus contain an antigenbinding site capable of immunoreacting with a particular epitope of theantigen characterized by a unique binding affinity for it.

As used herein, the term “KIM-1 proteolysis-inhibiting antibody” refersto an antibody that inhibits proteolytic release of a soluble KIM-1polypeptide.

As used herein, the term “crossblocking antibody” refers to an antibodythat lowers the amount of binding of anti-KIM-1 antibody to an epitopeon a KIM-1 polypeptide relative to the amount of binding of theanti-KIM-1 antibody to the epitope in the absence of the antibody.

As used herein, the term “conjugate” refers to an antibody covalentlylinked to a second moiety. The second moiety can be, e.g., a label.

As used herein, the term “label” refers to a molecular moiety capable ofdetection. A label can be, e.g., a radioactive isotope, enzyme,luminescent agent, or a dye.

As used herein, the term “fusion polypeptide” refers to an anti-KIM-1antibody molecule operatively linked to a non-anti-KIM-1 antibodymolecule.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration showing the polypeptide sequence ofthe human KIM-1 mucin domain (SEQ ID NO: 6) and corresponding 18-meroverlapping synthetic peptides used to map the binding epitopes ofmonoclonal antibodies ACA12, AKG7 and ABE3.

FIG. 1B is a set of graphs showing binding of monoclonal antibodiesABE3, AKG7 and ACA12 to peptides 43 to 50 at various concentrations ofantibody (peptide 43 corresponds to amino acids 210–227 of SEQ ID NO:7;peptide 44 corresponds to amino acids 219–236 of SEQ ID NO:7; peptide 45corresponds to amino acids 228–245 of SEQ ID NO:7: peptide 46corresponds to amino acids 237–254 of SEQ ID NO:7; peptide 47corresponds to amino acids 246–263 of SEQ ID NO:7; peptide 48corresponds to amino acids 255–272 of SEQ ID NO:7; peptide 49corresponds to amino acids 264–281 of SEQ ID NO:7; and peptide 50corresponds to amino acids 273–290 of SEQ ID NO:7).

FIGS. 2A–3C are representations of Western blot analysis of cell reactedwith AGE3 (FIG. 2A), cell extracts reacted with rabbit pilyclonalanibodies against the human KIM-1(b) C-terminus (FIG. 2B), andconditioned media reacted with monoclonal antigody AKG7(FIG. 2C).

FIGS. 3A–3C are representations of Western blot analyses of COS-7 cellextracts or conditioned media reacted with AKG7 (FIG. 3A), ABE3 (FIG.3B), or rabbit polyclonal antibodies raised against the human KIM-1(b)C-terminus (FIG. 3C).

FIG. 4 is a histogram showing the concentration of soluble KIM-1 inconditioned medium of COS-7 cells expressing KIM-1(b) and grown in thepresence of different concentrations of ABE3 or a control murine IgG.

FIG. 5 is a graph showing the concentration of soluble KIM-1 in theconditioned medium of 769-P cells grown in the presence of differentconcentrations of BB-94 or GM6001 MMP inhibitors.

FIG. 6A is a sequence of human KIM-1(a) (SEQ ID NO: 7) and human HAVcr-1or KIM-1(b) (SEQ ID NO: 8). A single sequence is represented up toresidue 323 corresponding to the sequence common to the twopolypeptides. Underlined are the putative signal sequence andtransmembrane domain. Shaded are the four putative N-glycosylationmotifs. Italicized is the sequence of the synthetic peptide used toraise antibodies against the C-terminus of KIM-1(b).

FIG. 6B is a schematic representation of the KIM-1(b) protein. Greyboxes represent the signal sequence and the transmembrane domain.Cysteine residue in the Ig-like domain are marked (C). The fourtriangles represent putative N-glycans. The TSP-rich region is thickenedto schematize the mucin-like domain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features antibodies that bind specifically to aKIM-1 polypeptide and inhibit proteolytic release of a soluble form ofKIM-1 from KIM-1 expressing cells.

KIM-1 is one of a number of membrane proteins that also exist in asoluble (truncated) form. Although these soluble forms can result fromalternative splicing, they more often derive from proteolysis of themembrane form. The cleavage occurs close to the transmembrane domain,resulting in the release of physiologically active protein.

The antibodies described herein can be used to detect a cell expressinga KIM-1 polypeptide, e.g., a kidney cell. Because KIM-1 polypeptides areexpressed at high levels in post-ischemic or diseased kidney cells, theantibodies disclosed herein are useful for detecting injured or diseasedkidney cells in a subject. The antibodies can also be used to inhibitproteolytic cleavage of a KIM-1 polypeptide, and thereby inhibitfunctions or processes mediated by soluble forms of a KIM-1 polypeptide.The antibodies can also be administered to a subject to treat or preventrenal disease or injury in a subject.

Anti-KIM-1 Antibodies that Inhibit Proteolytic Release of a SolubleKIM-1 Polypeptide

To prepare proteolysis-inhibiting KIM-1 antibodies, immunogens are usedthat include the extracellular domain of a KIM-1 polypeptide. Theextracellular domain extends from amino acids 1 to 290 of the humanKIM-1 polypeptide. The amino acid sequences of human and rat KIM-1polypeptides, and the nucleic acids encoding the polypeptides, areprovided in WO97/44460, published Nov. 27, 1997, and in Ichimura et al.(1998) J. Biol. Chem. 273:4135–42.

Suitable antibodies include, e.g., polyclonal, monoclonal, chimeric,single chain, F_(ab), F_(ab′), F_(sc), F_(v), and F_((ab′)2) fragments,and an F_(ab) expression library. In general, an antibody moleculeobtained from humans can be classified in one of the immunoglobulinclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.Reference herein to antibodies includes a reference to all such classes,subclasses and types of human antibody species.

The extracellular domain of the KIM-1 polypeptide, or a portion orfragment thereof, can serve as an antigen, and additionally can be usedas an immunogen to generate antibodies that immunospecifically bind theantigen, using standard techniques for polyclonal and monoclonalantibody preparation. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, or at least 15 amino acid residues, or atleast 20 amino acid residues, or at least 30 amino acid residues. Apreferred anti-KIM-1 antibody binds to an epitope within, overlapping,or in close proximity to the amino acid sequence SSDGLWNNNQTQLFLEHS (SEQID NO: 1) in KIM-1. The results obtained in Example 2 suggested that abinding epitope of the anti-KIM-1 antibody ABE3 to the KIM-1 polypeptideis around the portion of the amino acid sequence of KIM-1 polypeptidegiven by SEQ ID NO: 1.

Various procedures known in the art may be used for the production ofpolyclonal or monoclonal antibodies that inhibit proteolytic release ofa soluble KIM-1 polypeptide from KIM-1-expressing cells. See, forexample, ANTIBODIES: A LABORATORY MANUAL, Harlow and Lane (1988) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Some of theseantibodies are discussed below. Antibodies generated against the KIM-1polypeptide can be characterized to identify their binding epitopesusing methods known in the art, including those described in Example 2,below. Antibodies can be screened to identify those that inhibitproteolytic release of soluble forms of KIM-1 using methods such asthose described in Example 4, below.

Some proteolysis-inhibiting antibodies have the same epitope specificityas the antibody produced by the hybridoma producing monoclonal antibodyABE3. Also contemplated are antibodies that crossblock binding of themonoclonal antibody ABE3 to an epitope present on a KIM-1 polypeptide.Crossblocking antibodies can be identified by comparing the binding ofthe monoclonal antibody ABE3 to a KIM-1 polypeptide in the presence andabsence of a test antibody. Decreased binding of the ABE3 monoclonalantibody in the presence of the test antibody as compared to binding ofthe ABE3 monoclonal antibody in the absence of the test antibodyindicates the test antibody is a crossblocking antibody.

Polyclonal Antibodies

For the production of polyclonal antibodies, any suitable animal (e.g.,rabbit, goat, mouse or other mammal) may be immunized by one or moreinjections of a polypeptide that includes the KIM-1 ectodomain. Thepolypeptide can be, for example, the naturally occurring KIM-1, achemically synthesized polypeptide representing the ectodomain, or arecombinantly expressed fusion protein. The fusion moiety or achemically conjugated moiety can be a second protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor.

The preparation can further include an adjuvant. Various adjuvants usedto increase the immunological response include, but are not limited to,Freund's (complete and incomplete), mineral gels (e.g., aluminumhydroxide), surface active substances (e.g., lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.),adjuvants usable in humans such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. Additionalexamples of adjuvants which can be employed include MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenicprotein can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by Wilkinson. Wilkinson(2000) The Scientist 14: 25–28.

Monoclonal Antibodies and Hybridomas

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell. Goding, MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59–103.Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor (1984) J. Immunol. 133:3001; Brodeur etal., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, MarcelDekker, Inc., N.Y., (1987) pp. 51–63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard (1980) Anal. Biochem. 107:220. Preferably, antibodies having ahigh degree of specificity and a high binding affinity for the targetantigen are isolated.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium. Alternatively, thehybridoma cells can be grown in vivo as ascites in a mammal.

A hybridoma that produces monoclonal antibody subclone ABE3.16 wasdeposited with the American Type Culture Collection at 10801 UniversityBoulevard, Manassas, VA 20110-2209 (U.S.A.) on May 2, 2001, and has beenassigned Accession Number PTA-3350. ABE3.16 is a subclone of ABE3.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of interest can be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat bind specifically to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells of the invention serve as apreferred source of such DNA. Cloning of immunoglobulin variable regiongenes using the polymerase chain reaction (PCR) is an establishedtechnique. See, e.g., Kettleborough et al., 1993, Eur. J. Immunol.23:206–211. Once isolated, the DNA can be placed into expressionvectors, which are then transfected into host cells such as simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison(1994) Nature 368:812–13) or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

Humanized Antibodies

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Theseantibodies are suitable for administration to humans without causing astrong immune response by the human against the administeredimmunoglobulin. Humanized forms of antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that are principally comprised of the sequence of a humanimmunoglobulin, and contain minimal sequence derived from a non-humanimmunoglobulin. Humanization can be performed following the method ofWinter and co-workers (Jones et al. (1986) Nature 321:522–525; Riechmannet al. (1988) Nature 332:323–327; Verhoeyen et al. (1988) Science239:1534–1536), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta (1992) Curr. Op. Struct. Biol. 2:593–596).

Human Antibodies

Fully human antibodies relate to antibody molecules in which essentiallythe entire sequences of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor et al. (1983) Immunol. Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole et al. 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77–96). Human monoclonal antibodies may be produced byusing human hybridomas (see Cote, et al. (1983) Proc. Natl. Acad. Sci.USA 80: 2026–2030) or by transforming human B-cells with Epstein BarrVirus in vitro (see Cole, et al. (1985) In: MONOCLONAL ANTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc., pp. 77–96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. Hoogenboom and Winter(1991) J. Mol. Biol., 227:381; Marks et al. (1991) J. Mol. Biol.,222:581. Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10:779–783 (1992)); Lonberg et al. (Nature 368:856–859(1994)); Morrison (Nature 368:812–813 (1994)); Fishwild et al.,(NatureBiotechnology 14: 845–51 (1996)); Neuberger (Nature Biotechnology 14:826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13:65–93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. See, e.g., PCT publication WO94/02602. The endogenousgenes encoding the heavy and light immunoglobulin chains in the nonhumanhost have been incapacitated, and active loci encoding human heavy andlight chain immunoglobulins are inserted into the host's genome. Thehuman genes are incorporated, for example, using yeast artificialchromosomes containing the requisite human DNA segments. An animal whichprovides all the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a methodincluding deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

A method useful for producing an antibody of the invention, such as ahuman antibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain. Additional usefulprocedures, i.e., a method for identifying a clinically relevant epitopeon an immunogen, and a correlative method for selecting an antibody thatbinds immunospecifically to the relevant epitope with high affinity, aredisclosed in PCT publication WO 99/53049.

F_(ab) Fragments and Single Chain Antibodies

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein (see e.g., U.S. Pat. No. 4,946,778). Inaddition, methods can be adapted for the construction of Fab expressionlibraries (see e.g., Huse et al. (1989) Science 246:1275–1281) to allowrapid and effective identification of monoclonal F_(ab) fragments withthe desired specificity for a protein or derivatives, fragments, analogsor homologs thereof. Antibody fragments that contain the idiotypes to aprotein antigen may be produced by techniques known in the artincluding, but not limited to: (i) an F_((ab′)2) fragment produced bypepsin digestion of an antibody molecule; (ii) an F_(ab′) fragmentgenerated by reducing the disulfide bridges of an F_((ab′)2) fragment;(iii) an F_(ab) fragment generated by the treatment of the antibodymolecule with papain and a reducing agent and (iv) F_(v) fragments.

Immunoconjugates

The antibodies described herein can be conjugated to an agent such as achemotherapeutic agent, imaging agent, toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent can be made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., (1987) Science 238:1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

The antibodies described herein can also be labeled with an imagingreagent that produces a detectable signal. Imaging reagents andprocedures for labeling antibodies with such reagents are well known(see, e.g., Wensel and Meares, Radio Immunoimaging andRadioimmunotherapy, Elsevier, N.Y., (1983); Colcher et al., Meth.Enzymol. (1986) 121:802–16. The labeled antibody can be detected usingart-recognized techniques, including, e.g., radionuclear scanning (see,e.g., Bradwell et al., in Monoclonal Antibodies for Cancer Detection andTherapy, Baldwin et al. (eds.) pp. 65–85, Academic Press (1985)).

Pharmaceutical Compositions

The antibodies described herein can be administered to a mammaliansubject, e.g., a human, to image kidney cells or to treat kidneycell-associated disorders. The antibodies can be administered alone, orin a mixture. For example, the antibodies can be administered in thepresence of a pharmaceutically acceptable excipient or carrier, such asphysiological saline. The excipient or carrier is selected on the basisof the mode and route of administration. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences (E. W.Martin), and in the USP/NF (United States Pharmacopeia and the NationalFormulary). A pharmaceutical composition is formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include, e.g., intravenous, intradermal, subcutaneous,oral (e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, polypropyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents foradjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody, antibody derivative, or antigen-bindingpolypeptide of the invention. As used herein, “therapeutically effectiveamount” means an amount effective, at dosages, and for periods of timenecessary, to achieve the desired therapeutic result. A therapeuticallyeffective amount of the antibody, antibody derivative, orantigen-binding polypeptide can vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antibody, antibody derivative, or antigen-binding polypeptide toelicit a desired response in an individual. When a therapeuticallyeffective amount is administered, any toxic or detrimental effects ofthe antibody, antibody derivative, or antigen-binding polypeptide areoutweighed by the therapeutically beneficial effects. As used herein,“prophylactically effective amount” means an amount effective, atdosages, and for periods of time necessary, to achieve the desiredprophylactic result. Because a prophylactic dose is administered insubjects prior to onset of disease, the prophylactically effectiveamount typically is less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response,e.g., a therapeutic or prophylactic response. For example, in someembodiments of the invention a single bolus is administered. In otherembodiments, several divided doses are administered over time. The dosecan be reduced or increased proportionately, as indicated by theexigencies of the situation. It is advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. As used herein, “dosage unit form” meansphysically discrete units suitable as unitary dosages for the mammaliansubjects to be treated, with each containing a predetermined quantity ofactive ingredient calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1–100 mg/kg, preferably 0.5–50 mg/kg, more preferably1–20 mg/kg, and even more preferably 1–10 mg/kg. Dosage values may varywith the type and severity of the condition being treated. For anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions. It is to be understood that dosage ranges set forth hereinare exemplary only and are not intended to limit the scope of theclaimed invention.

Parenteral injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, according to U.S. Pat. No.3,710,795, incorporated herein by reference.

In general, a suitable subject is any mammal to which a KIM-1 antibodymay be administered. Subjects specifically intended for treatmentinclude humans, nonhuman primates, sheep, horses, cattle, goats, pigs,dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.

Renal conditions that may be beneficially treated include those in whichinhibition of release of soluble KIM-1 from a cell expressing a KIM-1cell surface protein can ameliorate the condition. Examples of suchconditions are renal cancer or renal injury, including renal cancerssuch as renal carcinomas. Other conditions include, e.g., renal failure,chronic renal failure, acute nephritis, nephritic syndrome, renal tubuledefects, kidney transplants, toxic injury, hypoxic injury, and trauma.Renal tubule defects include those of either hereditary or acquirednature, such as polycystic renal disease, medullary cystic disease, andmedullary sponge kidney.

Deposits

A hybridoma which produces the monoclonal antibody ABE3.16 has beendeposited with the American Type Culture Collection (ATCC) under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purpose of Patent Procedure on May 2,2001, and bears the accession number ATCC PTA-3350. Applicantsacknowledge their duty to replace the deposit should the depository beunable to furnish a sample when requested due to the condition of thedeposit before the end of the term of a patent issued hereon. Applicantsalso acknowledge their responsibility to notify the ATCC of the issuanceof such a patent, at which time the deposit will be made available tothe public. Prior to that time, the deposit will be made available tothe Commissioner of Patents under the terms of 37C.F.R. § 1.14 and 35U.S.C. § 112.

The invention is further illustrated by the following experimentalexamples. The examples are provided for illustration only, and are notto be construed as limiting the scope or content of the invention.

EXAMPLE 1 Generation of Anti-KIM-1 Monoclonal Antibodies

Monoclonal antibodies were generated against the extracellular domain ofa human KIM-1 polypeptide. A construct (KIM-1-Ig) was constructed inwhich the extracellular domain of human KIM-1 (residues 1–290) wasattached to the Fc portion of human IgG1 (hinge CH2+CH3 domains) andcloned into a mammalian expression plasmid pEAG347. A human KIM-1(b)full-length cDNA was obtained by RT-PCR using mRNA from the humancarcinoma cell line 769-P and primers based on the published DNAsequence (Feigelstock et al. (1998) J. Virol. 72:6621–28).

The sequence of the cDNA obtained was identical to that of the cDNAobtained from human kidney and liver. The pEAG347 expression plasmidcontains a tandem promoter (SV40 Early/Adenovirus Major late) forconstitutive expression and the DHFR gene for methotrexate selection ofstably expressing cell lines. Transfected CHO cell lines expressing thefusion proteins were selected, adapted in suspension, and grown infermentors.

Four mice were immunized with human KIM-1-Ig. The increase of theantibody titer against KIM-1 was monitored by performing enzyme-linkedimmunoabsorbance assays (ELISA). The ELISAs were performed in 96-wellplates (MaxiSorb, Nunc). Plates were coated by incubation overnight at4° C. with 100 μl of antigen or trapping antibody in 50 mM sodiumcarbonate, pH 9.6. Potential remaining adsorption sites were thenblocked with BSA by incubation for one hour at room temperature with 400μl PBS containing 1% BSA. Plates were washed four times with PBST aftereach reaction step. Horseradish peroxidase (HRP) conjugates were used assecondary detection reagents, and the color reaction was performed withtetramethylbenzidine.

The mouse showing the highest serological titer against KIM-1 wasidentified and boosted with KIM-1-Ig. The mouse was then sacrificed, andits spleen cells were fused with FL653 myeloma cells at a 1:6 ratio ofspleen cell per myeloma cell. The cell fusions were plated in 96 welltissue culture plates in selection media at a density of 10⁵ cells perwell, a density of 3.3×10⁴ cells per well, or a density of 1.1×10⁴ cellsper well. Wells positive for growth were screened by ELISA forexpression of antibody against human KIM-1, and subcloned. At the end ofthe selection, the ten clones showing the strongest binding wereretained and characterized by ELISA and western blot analysis. Theresults are shown below in Table 1.

TABLE 1 CLONE AUF1 ASG1 ACA12 ABE3 ATE11 AMC12 AKG7 BIE6 AWE2 ARD5Isotype Glk G2b Glk Glk Glk Glk Glk Glk Glk Glk ELISAon + + + + + + + + + + hKIM- 1-Ig ELISA on + + − − − + − + + + hKIM-1(mucin- minus)-Ig Western − − + + + − + − − − blot KIM-1-Ig reducedWestern + + + + + + + + + + blot KIM-1-Ig nonreduced

Four hybridoma clones (ABE3, ACA12, AKG7, and ARD5) were grown in theperitoneal cavity of mice. The ascitic fluid was collected, and eachantibody was purified by chromatography using protein A Sepharose.Biotinylated AKG7 was prepared by directed binding of amino-reactivesulfo-NHS-LC-biotin (Pierce).

EXAMPLE 2 Identification of Binding Epitopes of Anti-KIM-1 MonoclonalAntibodies

The monoclonal antibodies produced by hybridomas ABE3, ACA12, AKG7, andATE11 failed to bind to a KIM-1 fusion protein lacking the mucin domain(hKIM-1(mucin-min)-Ig). These results demonstrated that the bindingepitopes for these antibodies are at least partly in the mucin domain.In addition, these four antibodies were the only antibodies observed toreact with reduced denatured hKIM-1-Ig on a western blot. Thisobservation indicated that the epitopes for these antibodiescorresponded to a single stretch of amino acid residues in the proteinprimary structure.

Binding epitopes were identified by measuring binding of the antibodiesto eight overlapping synthetic 18-mer peptides starting at KIM-1 residue210, which lies within the mucin domain, and ending at residue 290, thelast residue of the extracellular domain. The peptides used in thebinding studies are presented in FIG. 1A relative to the KIM-1polypeptide sequence. The results of the binding studies are shown inFIG. 1B for monoclonal antibodies ACA12, AKG7, and ABE3. Both AKG7 andACA12 bound to peptides 45 and 46, although they differed in theirrespective binding affinities. From these results, it was concluded thatboth antibodies bound to a sequence including LQGAIRREP (SEQ ID NO:3),which is a 9-residue sequence common to peptide 45 and 46 (FIG. 1A).This peptide sequence lacked putative sites for either N-linked orO-linked glycosylation, which suggested that ACA12 and AKG7 recognizedboth glycosylated and non-glycosylated forms of the KIM-1 polypeptide.

Monoclonal antibody ABE3 bound to peptide #49 but did not showsubstantial binding to either peptide #48 or #50. These observationssuggested that the binding epitope of ABE3 is around DGLWNNNQTQL (aminoacids 3–13 of SEQ ID NO:1). This sequence includes a potentialglycosylation site at the last asparagine residue. However, since ABE3bound to synthetic peptides as well as to various glycosylated forms ofKIM-1, it was concluded that binding of ABE3 is primarily to a peptidemoiety.

EXAMPLE 3 Identification of a Shed form of a KIM-1 Polypeptide

This example demonstrated that soluble forms of KIM-1 polypeptides arereleased from KIM-1 expressing cells.

Three kidney and one liver human cell lines were analyzed by westernblot for expression of KIM-1. The cell lines used were 293 (embryonickidney cells transformed with adenovirus: CRL-1573), HK2 (human kidneyproximal tubular cells transducted with HPV-16; CRL-2190), 769-P (humanrenal cell adenocarcinoma; CRL-1933) and HepG2 (hepatocellularcarcinoma; HB-8065). Protein from cell extracts or conditioned mediawere analyzed by Western blot and probed with ABE3 monoclonal antibody,AKG7 monoclonal antibody, or a rabbit polyclonal antibody raised againstthe carboxyterminal portion of the KIM-1(b) protein. The rabbitpolyclonal antibody was raised against a synthetic peptide(CKEVQAEDNIYIENSLYATD (SEQ ID NO:4); Research Genetics) corresponding tothe last 19 amino acid residues at the C-terminus of the human KIM-1(b)protein, plus an additional cysteine residue for the conjugation. Thepeptide was conjugated to maleimide-activated KLH (Pierce) and theconjugate was used to immunize a rabbit. Antisera were collected afterseveral immunizations.

The conditioned medium and the cells were harvested at approximately 90%cell confluency. The cells were rinsed with PBS and scraped with arubber policeman in ice-cold PBS containing 5 mM EDTA and a cocktail ofprotease inhibitors (Boehringer Mannheim, Mini tablet). The cells werepelleted and lysed by resuspension in 50 mM HEPES, 150 mM NaCl, pH 7.5,1% NP-40 with protease inhibitors (20 μl of lysis solution per mg ofcell pellet). After 5 minutes on ice, the insoluble material wascollected by centrifugation for 5 minutes at 16000 g and the supernatantwas mixed with 2× reducing loading buffer. An aliquot of eachconditioned medium was also mixed with an equal volume of 2× reducingloading buffer.

For SDS-PAGE and western blot analysis, protein samples were mixed withreducing loading buffer and heated for 5 minutes at 95° C. Reduced anddenatured proteins were then separated by SDS-PAGE on 4–20%polyacrylamide gels. The proteins were transferred onto a nitrocellulosesheet. The blot was blocked with a solution of 5% non fat dry milk(Carnation) in PBST and probed in the same solution with the murinemonoclonal antibodies AKG7, ABE3 or ACA12 (at 1 μg/ml) or with therabbit polyclonal antiserum raised against the C-terminal peptide ofKIM-1(b) (diluted 1000 fold), followed by either goat anti-murine orgoat anti-rabbit antibodies conjugated to horse-radish peroxidase.Washes between the steps were performed with PBST. Reactive bands wererevealed by chemiluminescence.

Western blot analysis of the cellular extracts using ABE3 (FIG. 2A)revealed the expression of KIM-1 in the kidney carcinoma cell line (lane3) as well as in the transformed renal proximal tubule cell line HK2(lane 2); one major band at about 100 kDa as well as two other bands atabout 70 kDa and 50 kDa were detected. This pattern resembled theexpression pattern previously observed for the rat KIM-1 protein, whichalso appeared as three distinct bands after SDS-PAGE (Ichimura et al.,J. Biol. Chem. 273:4135–42). The same three bands were also observedwith a polyclonal antiserum raised against a synthetic peptidecorresponding to the C-terminus of human KIM1(b) (FIG. 2B).

Expression of KIM-1 could not be detected in the transformed embryonickidney cell line 293 or in the hepatocarcinoma cell line HepG2. Althoughthe expected size for the human KIM1 polypeptide is 36 kDa, the proteinband is expected to be detected at a much higher apparent molecularweight, as the protein presents four potential sites for N-glycosylationand multiple O-glycosylation sites.

Cell surface biotinylation of 769-P cells revealed that the 100 kDaKIM-1 band was the actual cell surface protein. The other bands mostlikely corresponded to KIM-1 processing intermediates transiting throughthe Golgi.

Western blot analysis of the conditioned culture media using AKG7monoclonal antibody (FIG. 2C) revealed the presence of a soluble KIM-1protein migrating at about 90 kDa. The 90 kDa band was also detectedwith ACA12 monoclonal antibody, but not with the ABE3 monoclonalantibody, or anti-KIM1(b) C-terminal antibodies. A protein band at about14 kDa was also detected with the anti-KIM1(b) C-terminus serum.Although this band could be unrelated to KIM-1, it is also possible thatthe 14 kDa band represents the C-terminal product of a cell surfacecleavage of KIM1(b).

The 10 kDa decrease in size, as well as the loss of ABE3 binding, couldbe the result of a cell surface proteolytic cleavage releasing a solubleform into the extracellular milieu. Alternatively, the soluble formcould arise from an alternative splicing event, in which the proteinproduced as a result of the alternative splicing lacked thetransmembrane and cytoplasmic domains.

To address the possibility that the 90 kDa soluble human KIM-1 formmight arise from alternative splicing of KIM-1 mRNA, recombinant humanKIM-1 was expressed from a KIM-1 cDNA construct (FIGS. 3A–3C). KIM-1(b)cDNA was cloned in a vector for constitutive overexpression (pEAG347) inmammalian cells. The resulting plasmid, phKIM1.2, and pEAG347 were usedto transfect COS-7 cells. Transformed cells were grown until theyreached confluency (about 4 days after transfection). Aliquots ofconditioned medium of KIM-1 expressing cells were taken after 1 and 2days. At day 4, the conditioned media were harvested and the cells wereprocessed as described in the previous section to obtain the cellularextract. The samples were analyzed by western blot with AKG7 (FIG. 3A),ABE3 (FIG. 3B) or polyclonal antibody raised against the carboxyterminus of hKIM1(b) (FIG. 3C).

Analogous to the pattern observed with native human KIM-1 from kidneycell lines, the recombinant KIM-1(b) appeared as several bandscorresponding to the various post-translationally modified forms of theprotein. Analysis of cell culture supernatant using AKG7 also showedvery clearly that a soluble form of KIM-1 was released and accumulatedin the cell culture medium. The released recombinant KIM-1, like solublenative KIM-1, was not detected with anti-h KIM1(b) C-terminalantibodies. It was also undetected with ABE3, although a faint band wassometimes observed at high concentration of the protein. Thisobservation suggested that part of the epitope was left after cleavageallowing some weak binding of the antibody. A 14 kDa band was alsodetected in the cellular extract exclusively with the anti-hKIM-1(b)-Cterminal polyclonal antibodies.

Together, these data revealed that soluble KIM-1 was released into theextracellular milieu by proteolytic cleavage at a site proximal to thetransmembrane domain, and overlapping with the ABE3 binding epitope.N-terminal sequencing analysis by Edman degradation of the soluble KIM-1isolated by immunoprecipitation and SDS□PAGE identified the sequenceSVKVGGEAGPXVXLX (SEQ ID NO:5) indicating that either the actual signalsequence is four residues longer than the predicted signal sequencedetermined by the method of von Heijne using the PSORT II program orthat the N-terminus is clipped after the removal of the signal peptide.

EXAMPLE 4 Inhibition of Shedding KIM-1 Polypeptide with ABE3 MonoclonalAntibody

To examine the effect of ABE3 monoclonal antibody on proteolytic releaseof KIM-1, COS-7 cells expressing transiently human KIM-1(b) were grownfor 2 days in the presence of various concentrations of ABE3 or mouseIgG as a negative control (FIG. 4).

For transient expression of human recombinant KIM-1(b), COS-7 cells weretransfected by electroporation with 10 μg of plasmid DNA per 10⁶ cells.Transfected cells were plated and grown in DMEM with 4 mM glutamine and10% fetal bovine serum. After 4 hours of incubation to allow the cellsto attach, the medium was replaced with fresh medium. Cell confluencywas then approximately 20%. For the shedding inhibition studies,transfected cells were plated in wells of 12-well plates and grown inmedium supplemented with various concentrations of ABE3 or mouse IgG(Sigma) as a negative control. Experiments were carried out intriplicates. After 2 days of incubation, the conditioned media werecollected from the wells, clarified by centrifugation and assayed byELISA using purified KIM-1-Ig as a standard.

The results are shown in FIG. 4. While the addition of non-specificmouse IgG did not affect the level of soluble KIM-1 in the medium, therewas a significant decrease of soluble KIM-1 when ABE3 was present in themedium. It is thus possible to inhibit partially the proteolytic releaseof the KIM-1 by competition binding with the ABE3 antibody. Thisconfirmed that the KIM-1 cleavage site lies at, or close to, the bindingsite of ABE3.

It is conceivable that the release of soluble KIM-1 could result from ahigh sensitivity of KIM-1 to non-specific proteases at the ABE3 bindingsite. However, cleavage was not observed of a C-terminus of arecombinant soluble KIM-1 corresponding to the entire extracellulardomain with 6 histidine residues at the C-terminus and expressedtransiently in the same medium as the full-length KIM-1.

EXAMPLE 5 Inhibition of KIM-1 Polypeptide Shedding withMetalloproteinase Inhibitors

Metalloproteinases (MMPs) or a desintegrin and metalloprotease (ADAMs)have been implicated in the specific cleavage of cell-surface proteins.The effect of two MMP inhibitors on cleavage of KIM-1 polypeptides wasexamined.

BB-94 (batismatat) and GM6001 (Ilomastat), are two broad spectrumhydroxamic acid-based zinc metalloproteinase inhibitors which inhibitseveral matrix metalloproteinases (MMP) (20). BB-94 is also a potentinhibitor of TACE (TNF alpha converting enzyme) (18). The activity ofthese two inhibitors on the shedding of KIM-1 in cell culture wasexamined.

Stock solutions of BB-94 and GM6001 were maintained in DMS0 at aconcentration of 70 mM for BB-94 and 2.5 mM for GM6001. Compounds werediluted directly into fresh medium to concentrations ranging from 0.5 μmto 32 μm. Renal carcinoma cells 769-P were grown in RPMI mediumsupplemented with 10% fetal bovine serum, 10 mM HEPES, and 1 mM sodiumpyruvate, and in the presence of various concentrations of BB-94 andGM6001 for 28 hours. The absence of cytotoxicity of the two compoundswas verified by checking cell viability at the end the 28h-cultureperiod using the mitochondria dye MTT (19)(21). The amount of solubleKIM-1 released into the extracellular milieu during the 28h-period wasmeasured by ELISA (FIG. 5).

A complete inhibition of KIM-1 shedding was achieved in the presence of32 μM of either MMP inhibitors. BB-94 appeared slightly more effectivewith an IC₅₀ of about 1 μM compared to GM 6001 for which the IC₅₀ wasabout 4 μM. These results indicated that the cleavage of KIM-1 ismediated by a metalloproteinase, possibly a member of the MMP or theADAM families.

Other embodiments are within the following claims.

1. An isolated monoclonal antibody or antigen-binding-fragment thereofthat binds to the extracellular domain of the human KIM-1 polypeptide ofSEQ ID NO:7 at an epitope within or overlapping the amino acid sequence:SSDGLWNNNQTQLFLEHS (SEQ ID NO:1).
 2. An isolated antibody orantigen-binding-fragment thereof that has the same epitope specificityas the antibody produced by the hybridoma deposited in the ATCC underAccession No. PTA-3350.
 3. An isolated antibody orantigen-binding-fragment thereof that crossblocks binding of theantibody produced by the hybridoma deposited in the ATCC under AccessionNo. PTA-3350.
 4. The isolated antibody or antigen-binding fragmentthereof of claim 1, wherein the monoclonal antibody is a humanizedantibody.
 5. The isolated antibody or antigen-binding-fragment thereofof claim 1, wherein the monoclonal antibody is a fully human antibody.6. A conjugate comprising the isolated antibody or antigen-bindingfragment thereof of claim 1, 2 or 3 linked to a detectable label.
 7. Aconjugate or fusion polypeptide comprising the isolated antibody orantigen-binding fragment thereof of claim 1, 2 or 3 and a toxin moiety.8. An antibody produced by the hybridoma deposited with ATCC underAccession No. PTA-3350.
 9. Hybridoma ABE3, deposited with ATCC underAccession No. PTA-3350.
 10. A composition comprising the isolatedantibody or antigen-binding fragment thereof of claim 1, 2 or 3 and apharmaceutically acceptable carrier.
 11. An isolated antibody orantigen-binding fragment thereof that binds to the extracellular domainof the human KIM-1 polypeptide of SEQ ID NO:7 at an epitope within theamino acid sequence: SSDGLWNNNQTQLFLEHS (SEQ ID NO: 1).
 12. The isolatedantibody or antigen-binding fragment thereof of claim 2, wherein theantibody is a humanized antibody.
 13. The isolated antibody orantigen-binding fragment thereof of claim 3, wherein the antibody is ahumanized antibody.
 14. The isolated antibody or antigen-bindingfragment thereof of claim 11, wherein the antibody is a humanizedantibody.
 15. The isolated antibody or antigen-binding fragment thereofof claim 2, wherein the antibody is a fully human antibody.
 16. Theisolated antibody or antigen-binding fragment thereof of claim 3,wherein the antibody is a fully human antibody.
 17. The isolatedantibody or antigen-binding fragment thereof of claim 11, wherein theantibody is a fully human antibody.
 18. The isolated antibody orantigen-binding fragment thereof of claim 2, wherein the antibody is amonoclonal antibody.
 19. The isolated antibody or antigen-bindingfragment thereof of claim 3, wherein the antibody is a monoclonalantibody.
 20. The isolated antibody or antigen-binding fragment thereofof claim 11, wherein the antibody is a monoclonal antibody.
 21. Theisolated antibody or antigen-binding fragment thereof of claim 1,wherein the monoclonal antibody is a single chain antibody.
 22. Theisolated antibody or antigen-binding fragment thereof of claim 2,wherein the antibody is a single chain antibody.
 23. The isolatedantibody or antigen-binding fragment thereof of claim 3, wherein theantibody is a single chain antibody.
 24. The isolated antibody orantigen-binding fragment thereof of claim 11, wherein the antibody is asingle chain antibody.
 25. The isolated antibody or antigen-bindingfragment thereof of claim 1, wherein the monoclonal antibody orantigen-binding fragment thereof is a chimeric antibody, an F_(ab)fragment, an F_((ab′)2) fragment, an F_(ab′) fragment, an F_(sc)fragment, or an F_(v) fragment.
 26. The isolated antibody orantigen-binding fragment thereof of claim 2, wherein the antibody orantigen-binding fragment thereof is a polyclonal antibody, a chimericantibody, an F_(ab) fragment, an F_((ab′)2) fragment, an F_(ab′)fragment, an F_(sc) fragment, or an F_(v) fragment.
 27. The isolatedantibody or antigen-binding fragment thereof of claim 3, wherein theantibody or antigen-binding fragment thereof is a polyclonal antibody, achimeric antibody, an F_(ab) fragment, an F_((ab′)2) fragment, anF_(ab′) fragment, an F_(sc) fragment, or an F_(v) fragment.
 28. Theisolated antibody or antigen-binding fragment thereof of claim 11,wherein the antibody or antigen-binding fragment thereof is a polyclonalantibody, a chimeric antibody, an F_(ab) fragment, an F_((ab′)2)fragment, an F_(ab′) fragment, an F_(sc) fragment, or an F_(v) fragment.29. An isolated antibody or antigen-binding-fragment thereof that bindsto the extracellular domain of the human KIM-1 polypeptide of SEQ IDNO:7, when expressed on the surface of a cell, at an epitope within oroverlapping the amino acid sequence: SSDGLWNNNQTQLFLEHS (SEQ ID NO:1).30. The isolated antibody or antigen-binding fragment thereof of claim29, wherein the antibody is a humanized antibody.
 31. The isolatedantibody or antigen-binding fragment thereof of claim 29, wherein theantibody is a fully human antibody.
 32. The isolated antibody orantigen-binding fragment thereof of claim 29, wherein the antibody is amonoclonal antibody.
 33. The isolated antibody or antigen-bindingfragment thereof of claim 29, wherein the antibody is a single chainantibody.
 34. The isolated antibody or antigen-binding fragment thereofof claim 29, wherein the antibody or antigen-binding fragment thereof isa polyclonal antibody, a chimeric antibody, an F_(ab) fragment, anF_((ab′)2) fragment, an F_(ab′) fragment, an F_(sc) fragment, or anF_(v) fragment.
 35. The isolated antibody or antigen-binding fragmentthereof of claim 29, wherein the antibody or antigen-binding fragmentthereof binds to an epitope within the amino acid sequence:SSDGLWNNNQTQLFLEHS (SEQ ID NO: 1).
 36. The isolated antibody orantigen-binding fragment thereof of claim 29, wherein the antibody is ahumanized antibody.
 37. The isolated antibody or antigen-bindingfragment thereof of claim 35, wherein the antibody is a fully humanantibody.
 38. The isolated antibody or antigen-binding fragment thereofof claim 35, wherein the antibody is a monoclonal antibody.
 39. Theisolated antibody or antigen-binding fragment thereof of claim 35,wherein the antibody is a single chain antibody.
 40. The isolatedantibody or antigen-binding fragment thereof of claim 35, wherein theantibody or antigen-binding fragment thereof is a polyclonal antibody, achimeric antibody, an F_(ab) fragment, an F_((ab′)2) fragment, anF_(ab′) fragment, an F_(sc) fragment, or an F_(v) fragment.